[unreadable] Neuropeptides influence mood, sensation, learning and memory, and the function of peripheral organs. Their release typically begins slowly only after bursts of electrical activity and is limited so that distal terminals are not easily emptied. Our goal is to understand how these unique properties are generated. Initially, we used live cell imaging of calcium and a GFP-tagged neuropeptide/hormone along with patch clamping to study peptide release by cultured cells. These experiments demonstrated great diversity in the handling of peptidergic vesicles. We then generated transgenic animals to study vesicle dynamics in synaptic boutons. In vivo experiments suggest that calcium influx through channels may increase vesicle motion within boutons to facilitate depletion of a diffuse pool of neuropeptidergic vesicles. Furthermore, we generated a new fluorescent protein construct to measure the delay in release following fusion pore formation. Finally, we discovered that a voltage-gated potassium channel that binds to secretory apparatus proteins can inhibit release without conducting ions. We are now poised to study three processes that could govern the time and activity dependence of neuropeptide release: DCV motion into and within synaptic boutons, delayed peptide dispersion following initial fusion, and voltage dependent channel interaction with the exocytosis machinery. Aim 1 will ascertain how DCVs move within boutons, and how neuropeptide release and DCV motion depend on electrical activity and intra-bouton calcium. Aim 2 will examine individual neuropeptide release events to determine whether peptidergic neurotransmission is sluggish because of postfusion events such as delayed peptide dispersion and kiss and run exocytosis. Aim 3 will determine whether the releasable DCVs in synapses are replenished first by refractory DCVs that were already present in boutons, or by new DCVs transported into the bouton from the axon. Aim 4 will test whether a potassium channel regulates release by changing the availability of t-SNARE proteins in a voltage-dependent manner. Understanding how channels, secretory vesicle dynamics and the release apparatus interact is essential for determining how neuropeptide release is uniquely controlled. This could provide a basis for controlling secretion of neuropeptides that are involved in pain, mood, hunger, and sleep. [unreadable] [unreadable]